1. Field of the Invention
The present invention relates generally to the fields of immunotherapy, oncology and infectious disease control. More particularly, it concerns novel methods of treating infectious diseases and cancers, in particular melanomas, with a combinatorial adjuvant or adjuvants in such a manner that an unexpectedly strong immune response directed against the diseases or melanomas is induced without the deleterious side effects that have been previously observed with standard chemotherapy.
2. Description of Related Art
Melanoma is a cancer of the pigmented cell of the skin, the melanocyte. Patients with metastatic (Stage IV) malignant melanoma have a median survival of approximately one year (Balch et al., 1993; Koh, 1991). Current standard treatment consists of combination chemotherapy with agents such as cisplatin, DTIC, and BCNU, with or without cytokines such as interleukin-2 (IL-2) or interferon-xcex1 (IFN-xcex1) (Balch et al., 1993; Koh, 1991; Legha and Buzaid, 1993). Response rates to chemotherapy have been reported to be as high as 60%, yet only approximately 5% of patients experience long term survival, regardless of the therapeutic regimen employed. Clearly, new approaches to the treatment of metastatic melanoma are needed.
Conventional chemotherapy aims to control the growth of cancer by targeting rapidly growing cells. However, this function is not specific, as many normal cells, such as those of the bone marrow and the intestinal epithelium, also have a basal level of proliferation. Therefore, many normal cells of the body also are susceptible to the toxic effects of chemotherapy, and conventional chemotherapy can impart a substantial degree of morbidity to the patient.
The attractiveness of immunotherapy is its specificity. If antigens were expressed on the tumor cells that were not expressed by normal cells of the host, then specific cytolytic T lymphocytes (CTL) could theoretically be activated to selectively kill the tumor cells while sparing the normal tissues of the patient. To this end, considerable effort has been made in the last decade to identify such tumor-specific antigens which may serve as targets for specific tumor cell killing (Boon et al., 1994; Boon et al., 1995).
Initial approaches to the immunotherapy of cancer have met with limited success. Vaccinations with irradiated tumor cells, with or without adjuvants, have generated response rates of 10-20% (Berd et al., 1990). Non-specific immune potentiators such as Bacillus Calmette-Guerin (BCG) also have given low but detectable response rates (Eilber et al., 1976). Treatment of patients with metastatic renal cell carcinoma with the T cell growth factor IL-2 has resulted in response rates of 15-20%, with several percent of patients experiencing a significant long-term survival (Hawkins, 1996). Similarly, the addition of IL-2 to standard chemotherapy for metastatic melanoma may result in increased response rates (Eilber et al., 1976). Collectively, these observations support the concept that immune manipulation has the potential to benefit patients with certain types of cancer, but clearly indicate that the current approaches are suboptimal. One hypothesis to explain the low overall response rates to these therapies is that the approaches up to now have aimed to amplify an immune response that has already been initiated by the host. In fact, the fundamental problem may be that most patients do not appropriately initiate an anti-tumor immune response at all. Further, tumor antigen-specific immunization will require induction of cytolytic T cell activity, and little is known regarding the optimal method of achieving this goal.
The molecular characterization of antigens specifically expressed on tumor cells but not on most normal cells of host origin has opened the possibility of tumor-specific vaccination in the immunotherapy of cancer. The last several years have witnessed a rapid expansion in the identification of human tumor antigens and their genes, chiefly in melanoma cell lines, that comprise several distinct categories: 1) point mutations in normal cellular genes; 2) differentiation antigens restricted to the melanocyte lineage; 3) intron sequences that become included in the coding region of a gene; 4) viral gene products; 5) underglycosylated normal gene products; and 6) developmentally regulated, non-mutated genes that. are not normally expressed in most adult tissues (Chen et al., 1993). Immune recognition of these antigens occurs via specific CD8+ CTL that interact with antigenic peptides bound to a groove in class I MHC (HLA) molecules. Class II MHC-binding epitopes recognized by CD4xe2x88x92 T cells also have been described.
Under optimal circumstances, initiation of an immune response is triggered by peptide/MHC complexes expressed by host antigen-presenting cells (APC), and additionally requires multiple cofactors provided by APC. Several cell types appear to be capable of serving as xe2x80x9cprofessionalxe2x80x9d APC, including dendritic cells (DC), activated B cells, and activated macrophages. After initial activation, CTL induced by APC interactions are thought to migrate throughout the host, recognize the same MHC/peptide complex on the tumor cells, and are triggered to kill them. This antigen-specific cytolysis is mediated largely via induction of apoptosis. It is hypothesized that one or several steps along this pathway of T cell activation and target cell recognition may be defective in tumor-bearing individuals.
MAGE-1 was the first human tumor antigen gene to be cloned and characterized (Van der Bruggen et al., 1991). It is expressed by several melanoma cell lines but not by any adult tissues except the testis. Therefore, it falls into category 6 listed above, being a normal gene that is abnormally expressed. MAGE-1 belongs to a family of at least 12 related genes, many of which also are expressed in various tumor cell types (De Plaen et al., 1994). One of these, MAGE-3, has been found to be expressed in approximately two-thirds of all melanoma samples tested. Peptides derived from the MAGE-3 protein have been identified that bind to the grooves of HLA-A1, HLA-A2, and HLA-B44 MHC molecules (Van der Bruggen et al., 1991; Van Pel et al., 1995; Van der Bruggen et al., 1994), and CTL recognizing each of these peptide/HLA combinations also have been observed. HLA-A2 is the most frequently expressed HLA allele in humans, present in about 50% of individuals.
Recently, 12 patients with MAGE-3+ metastatic melanoma were injected in Europe at monthly intervals with the MAGE-3 peptide that binds to HLA-A1 (Marchand et al., 1995). Either 100 or 300 xcexcg of peptide was injected in phosphate-buffered saline (PBS) at 2 subcutaneous sites distant from any tumor location. There were no major toxicities, and 3 patients experienced mild discomfort from inflammation at tumor locations or lymph nodes. Six patients were well enough to complete 3 monthly injections. Rather surprisingly, 3 of those 6 demonstrated major partial responses, giving an overall response rate of 25%.
Since the identification of the MAGE family, several additional melanoma antigens have been characterized including Melan-A, gp100 and tyrosinase (Old et al., 1996). One of these, Melan-A, is expressed by nearly all melanoma cell lines tested (Coulie et al., 1994), as well as in normal melanocytes. It therefore falls into category 2 above, encoding a melanocyte differentiation antigen. A peptide encoded by Melan-A has been defined that binds to HLA-A2.
Eighteen HLA-A2+ patients with metastatic melanoma were immunized with a peptide derived from Melan-A emulsified in incomplete Freund""s adjuvant (Cormier et al., 1997). No major toxicities were observed, and evidence of immunization was demonstrated in 12 patients. However, no tumor regression responses were seen, indicating that this rather straightforward vaccination strategy was not sufficient to generate a therapeutic effect. Collectively, these results support the general safety of tumor antigen peptides in humans, especially compared to the toxicities of conventional chemotherapy.
Recent advances in the understanding of T lymphocyte activation and differentiation have indicated several key costimulatory factors provided by APC that are vital for the optimal generation of CD8+ CTL. In fact, stimulation of T cells via the T cell receptor for antigen (TCR) in the absence of additional costimulatory factors has been shown to induce not activation, but rather an unresponsive state termed clonal anergy (Schwartz, 1990; Tan et al., 1993). Thus, participation of costimulator molecules is an essential component to initiating productive T cell differentiation. The specific cofactors present during and immediately after initial T-cell encounter with antigen determine the functional phenotype of the cells that emerge. For CD8+ T cells, the principal functional phenotypes fall into two subsets designated Tc1 and Tc2 (Sad et al, 1995). Tc1 cells produce high levels of IFN-xcex3 and TNF and have high lytic activity, whereas Tc2 cells produce IL4 and IL-5 and are poorly lytic (Cronin et al., 1995). It has been suggested that a Tc1-type response might be superior at mediating tumor rejection.
The B7-family of costimulator molecules, comprised of B7-1 and B7-2, appears to be important for instructing developing T cells to produce IL-2, and for preventing induction of T cell unresponsiveness or anergy (Linsley et al., 1991; Harding et al., 1992; Gimmi et al., 1993). B7-1/B7-2 interact with two counter-receptors, designated CD28 and CTLA4, on the surface of T lymphocytes. Provision of B7 during the activation of naive T cells is the trigger that gets the initial response going. At that point, the particular exogenous cytokines present determine the functional phenotype of the resulting activated effector cells. IL-12 appears to induce a high IFN-xcex3-producing Tc1 phenotype, whereas IL4 favors development of Tc2 cells (Sad et al., 1995). These characteristics parallel those of CD4+ helper T lymphocytes (Fitch et al., 1993). Provision of both B7 and IL-12 allows generation of potent tumor antigen-specific CTL in vitro (Gajewski et al, 1995). In several murine models in vivo, transfection of immunogenic tumors to express B7 has resulted in CD8+ T cell-dependent rejection by syngeneic mice (Townsend and Allison, 1993, Chen et al., 1994). IL-12 also can facilitate the regression of murine tumors in a T cell-dependent fashion (Brunda et al., 1993). Blockade of host B7 or IL-12 in vivo prevents the rejection of otherwise very immunogenic tumors (Gajewski et al., 1996; Fallarino et al., 1996), indicating that these two factors are normally employed by the immune response mediating tumor rejection.
The present invention provides methods of overcoming shortcomings of the prior art by providing improved methods of treating diseases and infections that are unexpectedly effective in inducing immune responses directed against diseases and infections. In some preferred embodiments the invention relates to treating melanomas, such as metatstatic melanomas, and viral infections. The inventors have discovered that administration of the adjuvants in the described manner are far more effective than would have been predicted based on the prior art or when the adjuvants are administered either alone or in a different combination or order. The invention provides the further advantage of reducing deleterious side effects that have been previously associated with cancer therapies.
As used in this specification and the appended claims and in accordance with long-standing patent law practice, the singular forms xe2x80x9caxe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d generally mean xe2x80x9cat least onexe2x80x9d, xe2x80x9cone or morexe2x80x9d, and other plural references unless the context clearly dictates otherwise. Thus, for example, references to xe2x80x9ca cellxe2x80x9d, xe2x80x9ca peptidexe2x80x9d and xe2x80x9can adjuvantxe2x80x9d include mixtures of cells, one or more peptides and a plurality of adjuvants of the type described; and reference to xe2x80x9cIL-12xe2x80x9d includes different species of such IL-12, for example, recombinant human IL-12, and so forth.
As used herein, the term xe2x80x9ca recombinant peptidexe2x80x9d, unless otherwise expressly stated, is used to succinctly refer to a recombinant peptide which is derived from an antigen that can be recognized by T-lymphocytes. xe2x80x9cRecombinant peptidesxe2x80x9d are generally peptide molecules that may be provided to cells (or animals) by the hand of man. The term xe2x80x9crecombinantxe2x80x9d peptide does not generally extend to amino acid sequence, peptides and proteins that may have been moved by a process of nature such that they have xe2x80x9crecombinedxe2x80x9d in a sequence or order different to the parent cell or organism from which they were derived without intervention by man.
The invention provides a method of inducing a mammalian immune response comprising: providing a composition comprising IL-12 and antigen-presenting cells pulsed with peptide and administering the composition to a mammal in an amount effective to induce an immune response. In one illustrative system the composition, or adjuvant, comprises peptide-pulsed, or loaded, antigen-presenting cells (APCs) and IL-12.
The invention further provides that the APCs comprise autologous cells and in some illustrative embodiments the antigen-presenting cells may comprise B cells activated by lipopolysaccharide, whole spleen cells, dendritic cells, fibroblasts or non-fractionated peripheral blood mononuclear cells (PMBC). Of course, it is understood that one of skill in the art will recognize that other antigen-presenting cells may be useful in the invention and that the invention is not limited to the exemplary cell types which are described herein.
The APCs are pulsed, or loaded, with antigenic peptide or recombinant peptide derived from at least one antigen. In one embodiment the peptide comprises an antigenic fragment capable of inducing an immune response that is characterized by the production of cytolytic T lymphocytes (cytolytic T cells or CTLs) which are directed against a malignancy or infection by a mammal. In a particular exemplary embodiment the peptide comprises one or more fragments of an antigen binding to class I MHC or class II MHC molecules (see Tables 1 and 2 for lists of exemplary tumor antigens). It is understood that the antigens listed in Tables 1 and 2 are provided for illustrative purposes and the skilled artisan will recognize that the described invention is not limited to these illustrative antigens.
In an illustrative system, the peptides comprise one or more fragments of one or more antigens expressed by melanoma tumors or other cancers, or infectious agents such as viruses, bacteria, parasites and the like. In some illustrative embodiments of the invention the peptide comprises MAGE-1, MAGE-3, Melan-A, P198, PIA, gp100 or tyrosinase. Of course, it is understood that one of skill in the art will recognize that peptides comprising one or more fragments of other antigens may be useful in the invention and that the invention is not limited to the exemplary peptides and antigens which are described herein.
APCs may be pulsed with any effective concentration of peptide. In a particular illustrative system, the APCs comprise cells pulsed with about 0.1 xcexcM-1 xcexcmM peptide. In a preferred illustrative system, the APCs comprise cells pulsed with about 1 xcexcM-100 xcexcM peptide, with a further preferred embodiment with about 10 xcexcM-50 xcexcM.
In a further embodiment the malignancy comprises a melanoma or other cancer, such as cancer of the prostate, ovary, kidney, lung, brain, breast, colon, bone, skin, testes or uterus, and the virus comprises a retrovirus, adenovirus, vaccinia virus, herpesvirus, adeno-associated virus, lentivirus, human immunodeficiency virus (HIV), or an arbovirus (arthropod-borne virus) (comprehensive lists and descriptions of arboviruses are provided in Entomology in Human and Animal Health, 7th ed., 1979 and The Biology of Disease Vectors, University Press Colorado, 1996, both of which are incorporated herein by reference). In another embodiment the infection comprises a bacterial or parasitic infection.
Mammals include, but are not limited to, equines, cattle, felines, canines, rats, mice and humans.
In a particular embodiment, the invention provides a method of inducing a mammalian immune response, wherein the peptide-pulsed APCs are administered to a mammal in need thereof, in a single therapeutic dose in combination with a single therapeutic dose of IL-12 followed by multiple therapeutic doses of IL-12.
Dosages may be any that induce an immune response. In certain embodiments, the amount of APCs administered comprises 1xc3x97106-1xc3x97109 per dose. In exemplary preferred embodiments the amount of APCs administered comprises about 1xc3x97108 per dose. In other embodiments the amount of IL-12 administered comprises 1 ng/kg-1000 ng/kg. In certain preferred exemplary embodiments the amount of IL-12 administered comprises 30-50 ng/kg per dose. Of course, it will be understood by the skilled artisan that the preferred dosage should be individualized to the patient following good laboratory practices and standard medical practices.
In another aspect the invention provides a method of treating a patient with a malignancy or infection comprising administering an adjuvant or composition comprising peptide-pulsed antigen-presenting cells and IL-12.
In a particular embodiment, the invention provides a composition using tumor antigen peptide pulsed autologous PBMC with and without rhIL-12 to produce an immune response in humans. In preferred embodiments, the tumor antigen peptide is Mage3 or MelanA. In further preferred embodiments, rhIL-12 is provided in addition to the Mage3 or MelanA.